Methods for the production of tyrosine, cinnamic acid and para-hydroxycinnamic acid

ABSTRACT

Genes encoding phenylalanine ammonia-lyase (PAL), tyrosine ammonia lyase (TAL) and phenylalanine hydroxylase (PAH) have been introduced into a host organism for the production of Para-hydroxycinnamic acid (PHCA). The introduction of these genes results in the redirection of carbon flow in the host, optimizing the flow of carbon from glucose to PHCA. The intermediates, tyrosine and cinnamic acid are also produced.

This application claims the benefit of U.S. Provisional Application No. 60/288,701, filed May 4, 2001.

FIELD OF THE INVENTION

The invention relates to the field of molecular biology and microbiology. More specifically, the invention relates to the production of tyrosine and para-hydroxycinnamic acid in a recombinant organism by the conversion of phenylalanine to tyrosine via phenylalanine hydroxylase and the subsequent conversion of tyrosine to para-hydroxycinnamic acid via tyrosine ammonium lyase.

BACKGROUND OF THE INVENTION

Production of chemicals from microorganisms has been an important application of biotechnology. Typically, the step in developing such a bio-production method may include 1) selection of a proper microorganism host, 2) elimination of metabolic pathways leading to by-products, 3) deregulation of such pathways at both enzyme activity level and the transcriptional level, and 4) overexpression of appropriate enzymes in the desired pathways. The present invention has employed combination of the steps above to redirect carbon flow from phenylalanine to tyrosine through phenylalanine hydroxylase which supplies the necessary precursor and energy for the desired biosynthesis of Para-hydroxycinnamic.

Para-hydroxycinnamic (PHCA) is a useful monomer for production of Liquid Crystal Polymers (LCP). LCP's may be used in electronic connectors and telecommunication and aerospace applications. LCP resistance to sterilizing radiation has also enabled these materials to be used in medical devices as well as chemical, and food packaging applications.

Para-hydroxycinnamic (PHCA) or p-coumarate is a known intermediate in the lignin biosynthetic pathway in plants (Plant Biochemistry, Ed. P. M. Dey, Academic Press, 1997). Methods of isolation and purification of PHCA are known (R. Benrief, et al., Phytochemistry, 1998, 47, 825–832; WO 972134). These methods are time consuming and cumbersome and a more facile method of production is needed for the large scale synthesis of this monomer. A fermentation route offers one possible solution.

A fermentation route to PHCA will require the engineering of several of the key enzymes involved in PHCA synthesis into an appropriate host. PHCA is a natural intermediate in the lignin biosynthetic pathway and key enzymes for synthesis may be obtained from a variety of plants. Lignin biosynthesis is initiated by the conversion of phenylalanine into cinnamate through the action of phenylalanine ammonia lyase (PAL). The second enzyme of the pathway is cinnamate-4-hydroxylase (C4H), a cytochrome P450-dependent monooxygenase (P450) which is responsible for the conversion of cinnamate to PHCA also called p-coumarate.

Thus, it is evident that one possible route to PHCA is via phenylalanine ammonia lyase (PAL) from phenylalanine. However this route also requires the presence of the second enzyme, cinnamate-4-hydroxylase, an enzyme which is rare in most microbes.

Information available indicates that PAL from some plants and micro-organisms can accept tyrosine as substrate in addition to its ability to convert phenylalanine to cinnamate. In such reactions the enzyme activity is designated tyrosine ammonia lyase (TAL). Conversion of tyrosine by TAL results in the direct formation of PHCA from tyrosine without the intermediacy of cinnamate. However, all natural PAL/TAL enzymes prefer to use phenylalanine rather than tyrosine as their substrate. The level of TAL activity is always lower than PAL activity, but the magnitude of this difference varies over a wide range. For example, the parsley enzyme has a K_(M) for phenylalanine of 15–25 μM and for tyrosine 2.0–8.0 mM with turnover numbers 22/sec and 0.3/sec respectively (Appert et al., Eur. J. Biochem. 225:491 (1994)). In contrast, the maize enzyme has a K_(M) for phenylalanine only fifteen times higher than for tyrosine, and turnover numbers about ten-fold higher (Havir et al., Plant Physiol. 48:130 (1971)). The exception to this rule, is the yeast, Rhodosporidium, in which a ratio of TAL catalytic activity to PAL catalytic activity is approximately 0.58 (Hanson and Havir in The Biochemistry of Plants; Academic: New York, 1981; Vol. 7, pp 577–625). Thus an alternate pathway to PHCA, might involve the direct conversion of tyrosine to PHCA via TAL, assuming an abundant source of tyrosine. Tyrosine is however, generally in low supply in most microorganisims, whereas phenylalanine is abundant. A method to convert phenylalanine to tyrosine would facilitate the pathway to PHCA through TAL.

Phenylalanine hydroxylase (PAH) systems appear to be infrequent in prokaryotes. Phenylalanine hydroxylase has been reported in a few species belonging to the α division of the class Proteobacteria and in Pseudomonas aeruginosa in the γ division (Zhao et al., Proc. Natl. Acad. Sci. USA. 91: 1366 (1994)). Of these, Pseudomonas aeruginosa is the best characterized at the molecular-genetic level (Song et al. Mol. Microbiol. 22:497–507 (1996) and Zhao et al., Proc. Natl. Acad. Sci. USA. 91: 1366 (1994)). Pseudomonas aeruginosa possesses a multi-gene operon that includes phenylalanine hydroxylase which is homologous with mammalian phenylalanine hydroxylase, tryptophan hydroxylase, and tyrosine hydroxylase (Zhao et al., Proc. Natl. Acad. Sci. USA. 91: 1366 (1994)). The bacterial Phenylalanine hydroxylase from Pseudomonas aeruginosa and Chromobacterium violaceum has been cloned, expressed, purified, and fully characterized (Abu-Omar et al. Book of Abstracts, 219 ACS National Meeting, San Francisco, Calif., Mar. 26–30, (2000) INOR-068 Publisher: American Chemical Society, Washington, D.C.)). Moreover, the presence of PAH in Streptomyces aureofaciens has been demonstrated (Maladkar, Hind. Antibiot. Bull., 28: 1–4, 30–6 (1986)).

The enzymatic conversion of phenylalanine to tyrosine is known in eukaryotes. Human phenylalanine hydroxylase is specifically expressed in the liver to convert L-phenylalanine to L-tyrosine (Wang et al. J. Biol. Chem. 269 (12): 9137–46 (1994)). Deficiency of the PAH enzyme causes classic phenylketonurea, a common genetic disorder.

The literature is silent as to the conversion of glucose to para-hydroxycinnamic acid via re-directing the carbon flow from phenylalanine to tyrosine through phenylalanine hydroxylase maximizing the concentration of tyrosine in the cells to allow for the use of the TAL pathway for conversion of glucose to PHCA.

The problem to be solved here is to develop an industrially suitable method for production of tyrosine and para-hydroxycinnamic acid using genetically engineered microorganisms.

Applicants have solved the stated problem by engineering several recombinant microorganisms comprising at least one gene encoding a phenylalanine hydroxylase activity and at least one gene encoding a phenylalanine and tyrosine ammonium lyase activity for the production of tyrosine and para-hydroxycinnamic acid from fermentable carbon substrates.

SUMMARY OF THE INVENTION

The invention provides a method for the production of para-hydroxycinnamic acid comprising:

a) providing a recombinant organism comprising:

-   -   i) at least one gene encoding a tyrosine ammonium lyase         activity; and     -   ii) at least one gene encoding a phenylalanine hydroxylase         activity;

b) growing said recombinant organism in the presence of a fermentable carbon substrate whereby para-hydroxycinnamic is produced.

Additionally the invention provides a method for the production of tyrosine comprising:

a) providing a recombinant organism comprising at least one gene encoding a phenylalanine hydroxylase activity; and

b) growing said recombinant organism in the presence of a fermentable carbon substrate whereby tyrosine is produced.

In another embodiment the invention provides recombinant hosts comprising:

i) at least one gene encoding a tyrosine ammonium lyase activity; and

ii) at least one gene encoding a phenylalanine hydroxylase activity.

BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS AND BIOLOGICAL DEPOSITS

The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions which form a part of this application.

The following sequences conform with 37 C.F.R. 1.821–1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO:1 is the nucleotide sequence encoding the C. violaceum Phenylalanine hydroxylase enzyme.

SEQ ID NO:2 is the deduced amino acid sequence of the C. violaceum Phenylalanine hydroxylase enzyme encoded by the nucleotide sequence of SEQ ID NO:1

SEQ ID NO:3 is the nucleotide sequence encoding the wildtype R. glutinis PAL enzyme.

SEQ ID NO:4 is the deduced amino acid sequence encoded by the nucleotide sequence encoding the wildtype R. glutinis PAL enzyme.

SEQ ID NO:5 and SEQ ID NO:6 are primers used for the isolation of the PAH gene of the Chromobacterium violaceum.

SEQ ID NOs:7–16 are primers designed and used for amplifying the PAH operon and its components in the E. coli hosts:

SEQ ID NOs:17–22 are mutant TAL proteins having amino acid substitutions within the wildtype R. glutinis PAL/TAL amino acid.

SEQ ID NO:23 is the nucleotide sequence encoding the mutant or modified R. glutinis PAL enzyme having enhanced TAL activity.

SEQ ID NO:24 is the deduced amino acid sequence encoded by the nucleotide sequence encoding the mutant R. glutinis PAL enzyme having enhanced TAL activity.

Applicants made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure:

International Depositor Identification Depository Reference Designation Date of Deposit P. aeruginosa containing the PTA 3349 May 2, 2001 modified PAL (SEQ ID NO:23) and the C. violaceum PAH (SEQ ID NO:1)

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes biological methods for the production of tyrosine, and PHCA. Furthermore, the present invention relates to the microorganisms which are genetically modified to increase carbon flow into the production of PHCA. Specifically, the present invention provides a method of producing tyrosine and PHCA in a phenylalanine producing microorganism using the phenylalanine hydroxylase gene of lactobacilli or bacilli to first convert phenylalanine to tyrosine.

In this disclosure, a number of terms and abbreviations are used. The following definitions are provided.

-   -   “Phenylalanine ammonia-lyase” is abbreviated PAL.     -   “Tyrosine ammonia lyase” is abbreviated TAL.     -   “Para-hydroxycinnamic acid” is abbreviated PHCA.     -   “Cinnamate 4-hydroxylase” is abbreviated C4H.     -   “Phenylalanine hydroxylase” is abbreviated PAH     -   “Phenylalanine 4-monooxygenase” is abbreviated phh A.     -   “4-alpha-carbinolamine dehydratase” is abbreviated phh B.     -   “Aromatic aminotransferase” is abbreviated phh C.     -   “4-alpha-carbinolamine dehydratase and aromatic         aminotransferase” is abbreviated phh BC.     -   “Phenylalanine 4-monooxygenase and 4alpha-carbinolamine         dehydratase” is abbreviated as phhAB     -   Phenylalanine 4-monooxygenase and aromatic aminotransferase is         abbreviated as phhAC     -   The term “Full operon” refers to a DNA fragment comprising phh         A, phh B and phhC genes

The term “TAL activity” or “TAL enzyme” refers to the ability of a protein to catalyze the direct conversion of tyrosine to Para-hydroxycinnamic acid (PHCA).

The term “PAL activity” or “PAL enzyme” refers to the ability of a protein to catalyze the conversion of phenylalanine to cinnamic acid.

The term “PAL/TAL activity” or “PAL/TAL enzyme” refers to a protein which contains both PAL and TAL activity. Such a protein has at least some specificity for both tyrosine and phenylalanine as an enzymatic substrate.

The term “modified PAL/TAL” or “mutant PAL/TAL” refers to a protein which has been derived from a wild type PAL enzyme which has greater TAL activity than PAL activity. As such, a modified PAL/TAL protein has a greater substrate specificity for tyrosine than for phenylalanine.

The term “PAH” activity” or “PAH enzyme” refers to a protein which catalyzes the conversion of phenylalanine to tyrosine.

As used herein the terms “cinnamic acid” and “cinnamate” are used interchangeably.

The term “fermentable carbon substrate” refers to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof.

The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the instant invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing as well as those substantially similar nucleic acid sequences.

“Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” or “wild type gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.

“Coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence.

“Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020).

“Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid”, “vector” and “cassette” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.

The term “amino acid” will refer to the basic chemical structural unit of a protein or polypeptide. The following abbreviations will be used herein to identify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Asparagine or aspartic acid Asx B Cysteine Cys C Glutamine Gln Q Glutamine acid Glu E Glutamine or glutamic acid Glx Z Glycine Gly G Histidine His H Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

The term “chemically equivalent amino acid” will refer to an amino acid that may be substituted for another in a given protein without altering the chemical or functional nature of that protein. For example, it is well known in the art that alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded protein are common. For the purposes of the present invention substitutions are defined as exchanges within one of the following five groups:

-   -   1. Small aliphatic, nonpolar or slightly polar residues: Ala,         Ser, Thr (Pro, Gly);     -   2. Polar, negatively charged residues and their amides: Asp,         Asn, Glu, Gln;     -   3. Polar, positively charged residues: His, Arg, Lys;     -   4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys);         and     -   5. Large aromatic residues: Phe, Tyr, Trp.

Thus, alanine, a hydrophobic amino acid, may be substituted by another less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine, or isoleucine). Similarly, changes which result in substitution of one negatively charged residue for another (such as aspartic acid for glutamic acid) or one positively charged residue for another (such as lysine for arginine) can also be expected to produce a functionally equivalent product. Additionally, in many cases, alterations of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W. Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. et al., In Current Protocols in Molecular Biology, published by Greene Publishing and Wiley-Interscience, 1987.

The present invention describes a method for the production of tyrosine as well as p-hydroxycinnamic acid in a recombinant microbial organism that produces phenylalanine. For the production of tyrosine the recombinant microorganism will contain a heterologus gene encoding a Phenylalanine hydroxylase (PAH) activity. For the production of p-hydroxycinnamic acid the recombinant organism will additionally contain a gene encoding a tyrosine ammonium lyase (TAL) activity. The relevant pathway is illustrated below:

The present method relies on a source of phenylalanine in the recombinant host. Phenylalanine may either be supplied exogenously or produced endogenously by the cell.

The invention is useful for the biological production of PHCA which may be used as a monomer for production of Liquid Crystal Polymers (LCP). LCP's may be used in electronic connectors and telecommunication and aerospace applications. LCP resistance to sterilizing radiation has also enabled these materials to be used in medical devices as well as chemical, and food packaging applications.

Additionally the invention provides a new method for the production of tyrosine, an amino acid used widely in industrial microbiology and in pharmaceutical synthetic methods.

Genes

The key enzymatic activities used in the present invention are encoded by a number of genes known in the art. The principal enzymes are phenylalanine hydroxylase (PAH) and tyrosine ammonium lyase (TAL).

Phenylalanine Hydroxylase (PAH) and Tyrosine Ammonium Lyase (TAL)

The invention provides a recombinant organism having PAH activity that is useful for the conversion of phenylalanine to tyrosine. This enzyme is well known in the art and has been reported in Proteobacteria (Zhao et al., In Proc. Natl. Acad. Sci. USA. 91: 1366 (1994)). For example Pseudomonas aeruginosa possesses a multi-gene operon that includes phenylalanine hydroxylase which is homologous with mammalian phenylalanine hydroxylase, tryptophan hydroxylase, and tyrosine hydroxylase (Zhao et al., In Proc. Natl. Acad. Sci. USA. 91: 1366 (1994

The enzymatic conversion of phenylalanine to tyrosine is known in eukaryotes. Human Phenylalanine hydroxylase is specifically expressed in the liver to convert L-phenylalanine to L-tyrosine (Wang et al. J. Biol. Chem. 269 (12): 9137–46 (1994)).

Although any gene encoding a PAH activity will be useful, and genes isolated from Proteobacteria will be particularly suitable, in the present invention it is preferred that genes encoding the PAH be isolated from Chromobacterium violaceum as set forth in SEQ ID NO:1.

In nature genes encoding phenylalanine ammonia-lyase are known to convert phenylalanine to cinnamate which may then be converted to Para-hydroxycinnamic acid via a p-450/p-450 reductase enzyme system. In many instances the phenylalanine ammonia-lyase has dual substrate specificity acting on phenylalanine principally, but also having some affinity for tyrosine. For example, the PAL enzyme isolated from parsley (Appert et al., Eur. J. Biochem. 225:491 (1994)) and corn ((Havir et al., Plant Physiol. 48:130 (1971)) both demonstrate the ability to use tyrosine as a substrate. Similarly, the PAL enzyme isolated from Rhodosporidium (Hodgins D S, J. Biol. Chem. 246:2977 (1971)) also may use tyrosine as a substrate. Such enzymes will be referred to herein as PAL/TAL enzymes or activities. Where it is desired to create a recombinant organism expressing a wild type gene encoding PAL/TAL activity, genes isolated from maize, wheat, parsley, Rhizoctonia solani, Rhodosporidium, Sporobolomyces pararoseus and Rhodosporidium may be used as discussed in Hanson and Havir, The Biochemistry of Plants; Academic: New York, 1981; Vol. 7, pp 577–625, where the genes from Rhodosporidium are preferred and the gene as set forth in SEQ ID NO:3 is most preferred.

In some instances it will be possible to increase the substrate specificity of the PAL/TAL enzyme via various forms of mutagenesis and protein engineering. A variety of approaches may be used for the mutagenesis of the PAL/TAL enzyme. Suitable approaches for mutagenesis include error-prone PCR (Leung et al., Techniques, 1:11–15 (1989) and Zhou et al., Nucleic Acids Res. 19:6052–6052 (1991) and Spee et al., Nucleic Acids Res. 21:777–778 (1993)) and in vivo mutagenesis. Protein engineering may be accomplished by the method commonly known as “gene shuffling” (U.S. Pat. No. 5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat. No. 5,830,721; and U.S. Pat. No. 5,837,458), or by rationale design based on three-dimensional structure and classical protein chemistry. Applicants have used a variety of these methods to determine which amino acid alterations within the classical Rhodosporidium PAL/TAL enzyme (SEQ ID NO:4) will give enhanced substrate specificity for tyrosine. These mutants, the altered amino acid residues, and the TAL/PAL activity are summarized below.

Strain Mutations TAL/PAL ratio Wild Type PAL None 0.5 EP18Km-6 CTG(Leu215) to CTC(Leu) 1.7 (mutant PAL GAA(Glu264) to GAG(Glu) GCT(Ala286) to GCA(Ala) ATC(lle540) to ACC(Thr) RM120-1 GAC(Asp126) to GGC(Gly) 7.2 CAG(Gln138) to CTG(Leu) CTG(Leu215) to CTC(Leu) GAA(Glu264) to GAG(Glu) GCT(Ala286) to GCA(Ala) ATC(lle540) to ACC(Thr) RM120-2 TTG(Leu176) to CTG(Leu) 2.1 GGC(Gly198) to CAC(Asp) CTG(Leu215) to CTC(Leu) GAA(Glu264) to GAG(Glu) GCT(Ala286) to GCA(Ala) ATC(lle540) to ACC(Thr) RM120-4 TCG(Ser181) to CCG(Pro) 2.0 GTC(Val235) to GCC(Ala) CTG(Leu215) to CTC(Leu) GAA(Glu264) to GAG(Glu) GCT(Ala286) to GCA(Ala) ATC(lle540) to ACC(Thr) RM120-7 TCG(Ser149) to CCG(Pro) 0.8 ATC(lle202) to GTC(Val) CTG(Leu215) to CTG(Leu) GAA(Glu264) to GAG(Glu) GGT(Ala286) to GCA(Ala) ATC(lle540) to ACC(Thr) RM492-1 GTC(Val502) to GGC(Gly) 2.0 CTG(Leu215) to CTC(Leu) GAA(Glu264) to GAG(Glu) GCT(Ala286) to GCA(Ala) ATC(lle540) to ACC(Thr)

It will be appreciated that the invention encompasses, not only the specific mutations described above, but also those that allow for the substitution of chemically equivalent amino acids. So for example where a substitution of an amino acid with the aliphatic, nonpolar amino acid alanine is made, it will be expected that the same site may be substituted with the chemically equivalent amino acid serine. Thus the invention provides mutant TAL proteins having the following amino acid substitutions within the wildtype PAL/TAL amino acid sequence (SEQ ID NO:4):

WT Sequence Amino ID No. Position Acid Possible Amino Acids 17 126 Asp Gly, Ala, Ser, Thr 138 Gln Leu, Met, Ile, Val, Cys 149 Ser Pro, Ala, Ser, Thr, Gly 181 Ser Pro, Ala, Ser, Thr, Gly 198 Gly Asp, Asn, Glu. Gln 202 Ile Val, Met, Leu, Cys 235 Val Ala, Gly, Ser, Thr, Pro 502 Val Gly, Ala, Ser, Thr, Pro 540 Ile Thr, Ala, Ser, Pro, Gly 18 126 Asp Gly, Ala, Ser, Thr 138 Gln Leu, Met, Ile, Val, Cys 540 Ile Thr, Ala, Ser, Pro, Gly 19 198 Gly Asp, Asn, Glu. Gln 540 Ile Thr, Ala, Ser, Pro, Gly 20 181 Ser Pro, Ala, Ser, Thr, Gly 235 Val Ala, Gly, Ser, Thr, Pro 540 Ile Thr, Ala, Ser, Pro, Gly 21 149 Ser Pro, Ala, Ser, Thr, Gly 202 Ile Val, Met, Leu, Cys 540 Ile Thr, Ala, Ser, Pro, Gly 22 502 Val Gly, Ala, Ser, Thr, Pro 540 Ile Thr, Ala, Ser, Pro, Gly

In addition to the rationale modifications recited above, mutations may be generated randomly. In the instant case a modified PAL enzyme was created from the wildtype R. glutinis PAL which had enhanced TAL activity. This mutation was created by error prone PCR and is set forth in SEQ ID NO:23, encoding a protein as set forth in SEQ ID NO:24.

It will be appreciated that the present invention is not limited to any specific sequence encoding either Phenylalanine hydroxylase or Tyrosine ammonia lyase activities but rather may be supplemented by genes having similar activities known in the art and obtainable by routine sequence dependant protocols. Examples of sequence-dependent protocols include, but limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of acid amplification technologies (e.g., polymerase chain reaction (PCR), ligase chain reaction (LCR)).

For example, genes encoding homologs of anyone of the mentioned activities (TAL, or PAH) could be isolated directly by using all or a portion of the known sequences as DNA hybridization probes to screen libraries from any desired plant, fungi, yeast, or bacteria using methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the literature nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primers DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of or full-length sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency. By these and other protocols a variety of genes encoding the Tyrosine ammonia lyase and phenylalanine hydroxylase activities may be isolated.

Microbial Hosts

The production organisms of the present invention will include any organism capable of expressing the genes required for PHCA or tyrosine production. Typically the production organism will be restricted to microorganisms.

Microorganisms useful in the present invention for the production of PHCA and tyrosine may include, but are not limited to bacteria, such as the enteric bacteria (Escherichia, and Salmonella for example) as well as Bacillus, Acinetobacter, Streptomyces, Methylobacter, Rhodococcus and Pseudomona; Cyanobacteria, such as Rhodobacter and Synechocystis; yeasts, such as Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia and Torulopsis; and filamentous fungi such as Aspergillus and Arthrobotrys, and algae for example. The PAL/TAL and PAH genes of the present invention may be produced in these and other microbial hosts to prepare large, commercially useful amounts of PHCA and tyrosine.

Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for production of PHCA. These chimeric genes could then be introduced into appropriate microorganisms via transformation to allow for expression of high level of the enzymes.

Vectors or cassettes useful for the transformation of suitable microbial host cells are well known in the art. Typically the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene which harbors transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.

Initiation control regions or promoters, which are useful to drive expression of the relevant genes in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, trp, IP_(L), IP_(R), T7, tac, and trc (useful for expression in Escherichia coli).

Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is most preferred if included.

Where commercial production of PHCA or tyrosine is desired a variety of fermentation methodologies may be applied. For example, large scale production may be effected by both batch or continuous fermentation.

A classical batch fermentation is a closed system where the composition of the media is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the medium is inoculated with the desired microorganism or microorganisms and fermentation is permitted to occur adding nothing to the system. Typically, however, the concentration of the carbon source in a “batch” fermentation is limited and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in the log phase generally are responsible for the bulk of production of end product or intermediate.

A variation on the standard batch system is the Fed-Batch system. Fed-Batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO₂. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Brock, T. D.; Biotechnology: A Textbook of Industrial Microbiology, 2nd ed.; Sinauer Associates: Sunderland, Mass., 1989; or Deshpande, M. V. Appl. Biochem. Biotechnol. 36:227, (1992), herein incorporated by reference.

Commercial production of PHCA or tyrosine may also be accomplished with continuous fermentation. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in their log phase of growth.

Continuous fermentation allows for modulation of any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by the medium turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium removal must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.

Production of PHCA or tyrosine will require suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose, raffinose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Additionally the carbon substrate may also be one-carbon substrates such as carbon dioxide, formaldehyde, formate or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated.

Description of the Preferred Embodiments

The PAH gene of the Chromobacterium violaceum (SEQ ID NO:1) was cloned and expressed in both Pseudomonas aeruginosa and DH5α E. coli. Production of tyrosine, cinnamate, and PHCA by Pseudomonas aeruginosa strains containing the PAH gene of Chromobacterium violaceum (SEQ ID NO:1) was demonstrated. Pseudomonas aeruginosa was demonstrated to lack p-450/p-450 reductase enzyme system needed to convert cinnamate to Para-hydroxycinnamic acid and thus all the Para-hydroxycinnamic acid produced in these experiments resulted from the re-direction of carbon flow through phenylalanine to tyrosine and then to Para-hydroxycinnamic acid. The production of cinnamate is likely a byproduct of the phenylalanine ammonia-lyase activity retained by the PAL/TAL enzyme.

In a particular application of the methods of the instant invention, it has been found that transformants of P. aeruginosa containing the PAH operon for conversion of phenylalanine to tyrosine with the modified PAL/TAL gene (SEQ ID NO:23) had an increased flow of carbon to tyrosine and an increased amount PHCA produced.

In a preferred embodiment, incorporation of C. violaceum PAH gene in combination with the expression of the endogenous P. aeruginosa PAH operon and the modified PAL/TAL resulted in increased amount of tyrosine and PHCA produced.

In another embodiment, P. aeruginosa PAH was cloned and expressed in the phenylalanine over-producing Escherichia coli and E. coli auxotrphic strain.

EXAMPLES

The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified. The meaning of abbreviations is as follows: “sec” means second(s), “min” means minute(s), “h” means hour(s), “psi” means pounds per square inch, “nm” means nanometers, “d” means day(s), “μL” means microliter, “mL” means milliliters, “L” means liters, “mm” means millimeters, “nm” means nanometers, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmole” mean micromole”, “g” means gram, “μg” means microgram and “ng” means nanogram, “U” means units, “mU” means milliunits and “U mg⁻¹” means units per mg.

Description of Strains:

Pseudomonas aeruginosa ATCC 15691 was used throughout as a control. Strain ATCC 15691 contains a native PAH operon, but lacks any PAL or TAL enzymatic functions and lacks the necessary p-450/p-450 reductase enzyme system to convert cinnamate to Para-hydroxycinnamic acid. Pseudomonas aeruginosa ATCC 15691 was also transformed with the native PAL/TAL from R. glutinis giving it the ability to convert phenylalanine to cinnamate. Additionally the Pseudomonas aeruginosa ATCC15691 was transformed with a mutant PAL/TAL enzyme [SEQ ID NO:23] (based on the R. glutinis wildtype sequence, SEQ ID NO:4) having enhanced substrate specificity for tyrosine. Additionally a Pseudomonas aeruginosa ATCC 15691 transformant was created containing a gene encoding the PAH from C. violaceum (SEQ ID NO:1) and a gene encoding the wildtype PAL from R. glutinis (SEQ ID NO:3). In similar fashion Pseudomonas aeruginosa ATCC 15691 was transformed with a gene encoding mutant PAL/TAL from R. glutinis [SEQ ID NO:23] having enhanced TAL activity and a gene encoding the PAH from C. violaceum (SEQ ID NO:1). In addition to Pseudomonas aeruginosa ATCC 15691, E. coli phenylalanine over producers were transformed with mutant and wildtype PAL/TAL genes, and PAH genes from C. violaceum.

The strains of the present invention are labeled in the following examples as follows:

-   -   Control=Pseudomonas aeruginosa (ATCC 15691)     -   TAL=Pseudomonas aeruginosa (ATCC15691) containing the modified         PAL/TAL (SEQ ID NO:23) from R. glutinis     -   PAH/TAL=Pseudomonas aeruginosa (ATCC 15691) containing the         modified PAL/TAL (SEQ ID NO:23) from R. glutinis and the PAH         (SEQ ID NO:1) from C. violaceum         Electroporation of Pseudomonas aeruginosa (ATCC 15691):

Samples of glycerol stocks of P. aeruginosa were spread onto agarose plates without any antibiotics and incubated overnight at 37° C. when colonies were at least 1.0 mm in diameter and dense. Cells (˜1.0–3.0 mg) were harvested from the plates and transferred to a tube using a sterile inoculating loop. Three generous swipes across a plate were usually satisfactory for obtaining the required samples. Collected cells were then resuspended, washed once in sterile water and resuspended in water (500 μl). Plasmid DNA (˜50 ng, plasmids described below) was added to the tubes containing the cells. The DNA/cell suspensions were then transferred to pre-chilled electorporation cuvettes (0.1 cm, Bio-Rad, Hercules, Calif.) and the cuvettes were kept on ice. Each sample was electrically pulsed at 18 kV/cm in a Gene Pulser (Bio-Rad) (25 μF, 200 om). SOC medium (1.0 ml) was added to each cuvette immediately after pulsing. The cell mixtures were then transferred to tubes and left on the shaker (1.0 hr at 37° C., 220 rpm). Samples (100 μl) of each transformation reaction were then pipetted onto separate LB plates and incubated overnight at 37° C.

Enzyme Activity Assay

The PAL or TAL activity of the purified enzymes was measured using a spectrophotometer according to Abell et al., “Phenylalanine Ammonia-lyase from Yeast Rhodotorula glutinis,” Methods Enzymol. 142:242–248 (1987). The spectrophotometric assay for PAL determination was initiated by the addition of the enzyme to a solution containing 1.0 mM L-phenylalanine and 50 mM Tris-HCl (pH 8.5). The reaction was then followed by monitoring the absorbance of the product, cinnamic acid, at 290 nm using a molar extinction coefficient of 9000 cm⁻¹. The assay was run over a 5 min period using an amount of enzyme that produced absorbance changes in the range of 0.0075 to 0.018/min. One unit of activity indicated deamination of 1.0 μmol of phenylalanine to cinnamic acid per minute. The TAL activity was similarly measured using tyrosine in the reaction solution. The absorbance of the para-hydroxycinnamic acid produced was followed at 315 nm and the activity was determined using an extinction coefficient of 10,000 cm⁻¹ for PHCA. One unit of activity indicated deamination of 1.0 μmol of tyrosine to para-hydroxycinnamic acid per minute.

SDS Gel Electrophoresis

The 8–25% native PhastGels were run with 4.0 μg of protein per lane and stained with Coomassie blue. Pharmacia High Molecular Weight (HMW) markers and grade I PAL from Sigma were used as standards.

Sample Preparation for HPLC Analysis

An HPLC assay was developed for measuring the levels of cinnamic acid and PHCA formed by the whole cells. In a typical assay, following centrifugation of a culture grown in the medium of choice, 20–1000 μL of the supernatant was acidified with phosphoric acid, filtered through a 0.2 or 0.45 micron filter and analyzed by the HPLC to determine the concentration of PHCA and cinnamic acid in the growth medium. Alternatively, following centrifugation, the cells were resuspended in 100 mM Tris-HCl (pH 8.5) containing 1.0 mM tyrosine or 1.0 mM phenylalanine and incubated at room temperature for 1.0–16 h. A filtered aliquot (20–1000 μL) of this suspension was then analyzed.

The HPLC Method:

A Hewlett Packard 1090M HPLC system with an auto sampler and a diode array UV/vis detector was used with a reverse-phase Zorbax SB-C8 column (4.6 mm×250 mm) supplied by MAC-MOD Analytical Inc. Flow rate of 1.0 mL per min, at column temperature of 40° C. was carried out. The UV detector was set to monitor the eluant at 250, 230, 270, 290 and 310 nm wavelengths.

Solvents/Gradients: Solvent A Solvent B Time (min) Methanol 0.2% TFA 0.0 10% 90% 0.1 10% 90% 9.0 36% 65% 9.1 50% 50% 14.0 50% 50% 18.0 0% 0% 21.0 0% 0%

Retention time (RT) of related metabolites using the HPLC system described above are summarized below.

Compounds (1.0 mM) RT (min)  1. tyrosine 6.7  2. phenylalanine 9.4  3. 4-hydroxybenzoic acid (PHBA) 11.6  4. 3,4-dihydroxycinnamate (caffeic acid) 12.5  5. 3-(4-hydroxyphenyl)propionate 13.3  6. 4-hydroxyphenylpyruvate 13.6  7. 4-hydroxyacetaphenone 14.0  8. 4-hydroxycinnamic acid (PHCA) 14.2  9. 2-hydroxycinnamic acid (OHCA) 15.3 10. benzoic acid 15.5 11. coumarin 16.0 12. cinnamyl alcohol 17.3 13. phenylpyruvate 18.1 14. cinnamic acid 18.3 MONO Q Buffer:

The buffer used for these analyses was a 50 mM potassium phosphate, pH 7.0, as the starting buffer followed by a 400 mM potassium phosphate buffer, pH 7.2 as eluent for the Mono-Q column.

Example 1 Cloning and Expression of Chromobacterium violaceum Phyenylalanine Hydroxylase in Microorganisms Phenylalanine Hydroxylase for Chromobacterium violaceum DNA Amplification and Cloning

In these studies the PAH gene of the Chromobacterium violaceum (SEQ ID NO:1) was cloned into both Pseudomonas aeruginosa (ATCC15691) and DH5α E. coli. Two oligonucleotide primers, 5′-TCCAGGAGCCCAGGATCCAACGATCGCGCCGA-3′ [SEQ ID NO:5], (designated CVPH168), and 5′-GGACAAGCTTAATGATGCAGCGACACAT-3′ [SEQ ID NO:6], (designated CVPH1170) were synthesized based on the deoxynucleotide sequences flanking the coding region of the C. violaceum phenylalanine hydroxylase gene described previously (A. Onishi, L. J. Liotta, S. J. Benkovic; Cloning and Expression of Chromobacterium violaceum phenylalanine hydroxylase in Escherichia coli and comparison of amino acid sequence with mammalian aromatic amino acid hydroylase J. Biol. Chem. 266:18454–18459 (1991)). Restriction endonuclease sites (BamHI or HindIII, underlined sequences of the above primers) were designed at the 5′-prime end of each primer to facilitate cloning. C. violaceum genomic DNA was isolated and purified with Qiagen Kit and the DNA amplification carried out. The amplification reaction mixture (100 μl) contained 1.0 mg of the genomic DNA template, 100 pmol each of the two primers, 2.5 units of Taq DNA polymerase (Qiagen) in 10 mM Tris-HCl, (pH 8.8), 0.2 mM each of the four dNTPs, 50 mM KCl, 1.5 mM MgCl2, and 0.01% bovine serum albumin. Thirty PCR cycles (94 C, 0.5 min; 55 C, 0.5 min and 72 C, 2.0 min) were performed. The correctly amplified DNA fragments (based on the fragment size) were cloned into a TA vector (Invitrogen, Carlsbad, Calif.) and submitted for sequence analysis. Several clones containing the gene were thus obtained.

Gene Expression and Protein Purification:

To express the cloned phenylalanine hydroxlyase gene, the amplified DNA fragments of interest were purified using a GeneClean II Kit (Bio 101, Inc, Carlsbad, Calif.), digested with endonuclease BamHI and HindIII (Promega, Madison, Wis.) and cloned into the BamHI and HindIII sites in plasmid vectors such as pET24a (Novogen, Madison, Wis.), pQE30 (Qiagen) and pTrc99A (Pharmacia, Piscataway, N.J.). The following designations were used for the vectors prepared: pETDW (PET24a vector containing PAH gene from C. violaceum (SEQ ID NO:1)). PQSW-PAH1170 (pQE30 vector containing the same gene) and pTrc-PAH (pTrc99a containing the PAH gene). The Pseudomonas and E. coli strains transformed electroporationally with the expression vectors were grown at 37° C. in LB medium containing either 25 mg/ml of kanamycin for vpESW-PAH1170 or 100 mg/ml of ampicillin for both pQSE-PAH1170 and pTrc-PAH in the absence or presence of isopropyl-beta-D-thiogalactoside (IPTG). Expression of the recombinant phenylalanine hydroxylase was analyzed by SDS-polyacrylamide gel electrophoresis. Purification of phenylalanine hydroxylase to homogeneity was accomplished by passing the cell suspensions in 50 mM acetate buffer pH 6.0 containing 1 μg/ml of leupeptin and pepstatin A through the French Pressure cell (×2) at 18,000 psi. Protease inhibitor, PMSF, was then added to the extract to a final concentration of 0.5 mM. Cell debris and inclusion bodies were removed by centrifugation at 38,000×g for 20 min. The supernatant was then used as the cell free extract.

Anion exchange was carried out on a 20 mm×165 mm, 50 μm HQ column at a flow of 30 ml/min. The starting buffer (buffer A) was 50 mM sodium acetate pH 6.0 containing 20 mM NaCl and the eluting buffer (buffer B) was 50 mM sodium acetate pH 6.0 containing 500 mM NaCl. The column was equilibrated and washed with buffer A after sample injection. A gradient was run from 100% of buffer A to 100% of buffer B over ten column volumes. The column was washed with buffer B and then re-equilibrated with buffer A. Protein was monitored at 280 nm and 10 ml fractions were collected after two column volumes of the gradient. All of the crude extract was used in one run on the column. Fractions containing PAH activity were pooled, and concentrated by Centricom-10 ultrafiltration. The purified enzyme was stored at −70° C. after being frozen in liquid nitrogen. The purity of the phenylalanine hydroxylase was judged by inspection of Coomassie Blue stained SDS polyacrylamide gels following electrophoresis of the samples.

TABLE 1 Purification of PAH from recombinant E. coli Total protein Concentration Total enzyme Purification Yield activity Specific act. Step (mg) (U)^(a) (U mg⁻¹) (fold)(%) Crude Extract 100 206 3481.5 16.93  1 Anion 47 1770.0 37.76 2.23 Exchange 51 Gel Filtration 32 1162.8 35.82 2.12 33 ^(a)One unit corresponds to 1 μmol of tyrosine produced per min. Determination of the Phenylalanine Hydroxylase Enzyme Activity and Protein Concentration:

The phenylalanine-dependent DMPH4 dehydrogenation activity was measured by monitoring tyrosine production spectrophotometrically. The assays were performed by the addition of the enzyme solution (1.0–10 ml) to a 1.0 ml solution containing L-phenylalanine (1.0 mM, 10 ml of a 100 mM solution); Dithiotreitol (DTT, 6.0 mM, 6.0 ml of a 1.0 M solution); Fe(NH₄)₂(SO₄)₂ (1.0 mM, 10 ml of a 100 mM solution), 6,7-dimtheyltetrahydropterin (DMPH4)(120 mM, 10 ml of a 12 mM solution)and HEPES buffer (960 ml of 100 mM, pH 7.4). The reaction was initiated by the addition of phenylalanine to the above mixture and followed by monitoring the increase at 275 nm due to the formation of the product, tyrosine. The assay was run for several minutes using an amount of enzyme that produced a change of absorbance around 0.01/min. Activities were determined using a molar extinction coefficient of 1700 cm⁻¹. One unit of enzyme activity will produce 1.0 umol of tyrosine per minute. The concentration of the enzyme in solutions was routinely determined by the BCA protein assay (Bio-Rad Laboratories, Hercules, Calif.).

In Vivo Production of Tyrosine:

Assays for in vivo tyrosine production were conducted by HPLC methods. The microorganisms containing the phenylalanine hydroxylase gene were inoculated into the LB medium with corresponding antibiotics in the presence or absence of cofactor DMPH4 and/or FeSO4. Cells were left on the shaker at 37° C. overnight. The cultures were then washed (×3) with M9 medium containing 2% glucose and resuspended in the same medium. Samples (1.0 ml) were taken at specified time intervals and prepared and analyzed by HPLC.

Example 2 Production of Tyrosine, Cinnamate and PHCA by Pseudomonas aeruginosa Strains Containing the Phenylalanine Hydroxylase Gene of Chromobacterium violaceum

This example describes expression of the phenylalanine hydroxylase from C. violaceum in Pseudomonas aeruginosa.

In order to confirm the source of Para-hydroxycinnamic acid produced in of P. aeruginosa (ATCC 15691) it was first necessary to confirm that of P. aeruginosa (ATCC 15691) did not have the enzymatic machinery to produce Para-hydroxycinnamic acid from cinnamate. To confirm this the following study was done.

A single colony was picked up from the LB agar plate and inoculated into 4.0 ml of the LB medium. The cultures were left on the shaker (30° C., 225 rpm) overnight. Then 200 ul of the culture was transferred into 150.0 ml of fresh LB medium and the cultures were left on the shaker (30° C., 225 rpm) overnight. Cells were then centrifuged and washed twice with the M9 medium containing 2% glucose. The cell pellet was then re-suspended into either the M9 medium, M9 medium containing 2% glucose, LB medium and LB medium containing 2% glucose and the OD at 600 nm was adjusted to 0.2. Either pHCA or cinnamate (1.0 mM) were added to the cultures which were then left on the shaker (30° C.). Cultures that did not receive either PHCA or cinnamate were used as Controls. Samples (1.0 ml) were taken from the of cultures at 24 hours and 48 hours for HPLC analysis. Results of the analyses are summarized below (Table 2) and indicate that P. aeruginosa could not produce PHCA from cinnamate and was not consumed in substantial amounts.

TABLE 2 Consumption of PHCA and Cinnamate by Wild Type P. aeruginosa (1.0 mM PHCA or 1.0 mM Cinnamate added) PHCA conc. (mM) Medium 24 hours 48 hours M9 medium with glucose 0.90 0.87 LB medium with glucose 0.95 0.98 M9 medium 0.95 0.91 LB medium 1.00 0.92

Cinnamate concentration. (mM) Medium 24 hours 48 hours M9 medium with glucose 0.87 0.85 LB medium with glucose 0.88 0.87 M9 medium 0.80 0.70 LB medium 0.95 0.84

The recombinant strains used in this study were prepared by electroporation of P. aeruginosa (ATCC 15691) with the expression vectors pE-PAL (containing the native PAL/TAL from Rhodotorula glutinis with Ampicillin selection marker) and pJ-PAH (containing PAH from Chromobacterium violaceum (SEQ ID NO:1) with Kanamycin selection marker). The double selection plate contained the LB medium plus Carbenicillin (175 μg/ml) and Kanamycin, (150 μg/ml). Single colonies were picked up from the selective plates and inoculated into tubes containing the LB medium (10 ml) with antibiotics. Cultures were left on the shaker overnight (37° C. and 225 rpm). They were then centrifuged, washed once with LB medium and resuspended in 10 ml of the same medium. Isopropyl beta-D-thiogalactoside (IPTG, 10 μl of a 1.0 M stock solution) was added to the cultures (final concentration 1.0 mM) and left on the shaker, (37° C., 225 rpm, 24 hr). Samples (1.0 ml) were taken after 24 hours of induction with IPTG and prepared for HPLC analysis to determine the concentrations of tyrosine, cinnamate and PHCA in them. Results of HPLC analysis are summarized in Table 3. The control cells (native P. aeruginosa containing the Pseudomonas PAH operon), and the PAL and TAL transformants did not produce any tyrosine following growth on the LB medium. However, Pseudomonas transformants containing the PAH from C. violaceum [SEQ ID NO:1] (in addition to their native PAH) and those transformants containing either the PAH/PAL or PAH/TAL produced 233.5, 13.6 and 23.5 M of tyrosine respectively under these experimental conditions. Analysis for cinnamic acid production by various strains indicated production of very low levels (1.45 and 12.2 M respectively) of cinnamate by both the native P. aeruginosa strain and the cells containing the PAH from C. violaceum [SEQ ID NO:1]. However cells containing either PAL or TAL showed much higher levels of cinnamate production (322.3 and 220.8 M respectively). Cells that contained C. violaceum PAH in addition to either PAL or TAL showed formation of lower amounts (204.8 and 109.5 m respectively) of cinnamate compared to those containing only PAL or TAL. Similarly only small amounts of PHCA (0.46 and 0.37 M respectively) was formed by the Control and the PAH containing cells while much higher levels of PHCA (188.9 and 285.1 M respectively) were observed with cells containing PAL and TAL or the combination of PAH/PAL and PAH/TAL (155.6 and 207.5 m respectively). These results clearly underline the effect of the PAH enzyme in diverting the flow of Carbon from phenylalanine to tyrosine as attested by observation of tyrosine in Pseudomonas recombinant strains containing the C. violaceum gene in addition to their native PAH operon. The fact that somewhat lower levels of PHCA was formed by transformants containing both PAH and PAL or TAL could reflect the rate limiting role of PAH in these cells.

TABLE 3 Tyrosine, Cinnamate and PHCA Production by P. aeruginosa Transformants Tyrosine PHCA Cinnamic Acid Transformants (μM) (μM) (μM) Control 0 0.46 1.45 PAL 0 188.9 322.3 TAL 0 285.1 220.8 PAH 233.5 0.37 12.2 PAH/PAL 13.6 155.6 204.8 PAH/TAL 23.5 207.5 109.5

Example 3 Production of Tyrosine, Cinnamic Acid and PHCA by Recombinant Pseudomonas aeruqinosa Strains Following Growth in M9 Plus Glucose Production of Tyrosine, Cinnamic acid and PHCA by Recombinant Pseudomonas aeruginosa Strains

Recombinant strains used in this study were those described in Example 1. Experimental procedure was similar to that of Example 1 with the exception that in this example, cells were grown on the defined M9 medium and then glucose was added as the sole source of carbon and the samples were taken for analysis after 6 hours instead of 24 hours. The change in the growth medium from LB (Example 2) to the defined M9 plus glucose (Example 3) resulted in a significant reduction in the cell mass produced and therefore the values obtained for the concentration of tyrosine, cinnamate and PHCA produced are significantly lower than those obtained in Example 1.

The results obtained indicated that the Control cells (although they already contained the native PAH operon) could not produce tyrosine from glucose. When these cells were transformed with modified PAL/TAL (SEQ ID NO:23), tyrosine (95.54 M) was observed in the 6 hour samples. However, these cells did not produce any cinnamate and/or PHCA. When TAL was incorporated into the Pseudomonas cells negligible amounts of tyrosine (3.44 M) were detected. Cinnamate and PHCA (18.49 M and 13.59 M respectively) were detected in these cultures. The transformants containing both PAH and TAL produced the highest levels of tyrosine, cinnamate and PHCA (97.24 M, 23.44 M, and 19.19 M respectively). Production of cinnamate by cells containing TAL and/or PAH/TAL confirms the results discussed in Example 1 that over-expression of the PAH in the Pseudomonas is required to allow for tyrosine production in the medium and that the TAL still contains some PAL activity.

TABLE 4 Tyrosine, PHCA, and Cinnamic Acid Production from Glucose by P. aeruginosa Transformants Product Production (μM) Transformants Tyrosine PHCA Cinnamic Acid Control 0 0 0 PAH 95.54 0 0 TAL 3.44 13.59 18.49 PAH/TAL 97.24 19.19 23.44

Example 4 Tyrosine and PHCA Production by P. aeruginosa TAL Transformants with or without Phenylalanine Addition

The P. aeruginosa contains the PAH operon for conversion of phenylalanine to tyrosine. Transformants of this strain were prepared with the modified PAL/TAL gene (SEQ ID NO:23) in order to study the effect of the P. aeruginosa's PAH operon on increasing the flow of carbon to tyrosine and therefore increasing the amount of PHCA produced. Transformants were grown in the LB medium and were then divided into two groups. To one group, phenylalanine (0.1 mM) was added while the other group did not receive any additional phenylalanine. Samples were taken after 6.0 hours and as can be seen in the Table 5. The level of tyrosine detected in the medium increased from ˜23 M in control uninduced cells to ˜66 M by the IPTG induced transformants without additional phenylalanine to ˜350 M by the induced transformants which had received additional phenylalanine. Control cells did not produce any PHCA while ˜10 and 13.0 M of PHCA was detected in the cultures without and with phenylalanine addition respectively.

TABLE 5 Tyrosine and PHCA Production by P. aeruginosam PAL/TAL Transformants with or without Phenylalanine Addition Product Production (μM) Sample Tyrosine PHCA Control (uninduced) 22.59 0 No Phenylalanine Addition 65.55 9.77 0.1 mM Phenylalanine Addition 350.61 13.72

Example 5 Tyrosine and PHCA Production by P. aeruqinosa PAH Transformants with or without Phenylalanine Addition

Experiments were performed using the P. aeruginosa transformed with the C. violaceum PAH (SEQ ID NO:1) to investigate their ability to produce tyrosine (Table 6). Cells were grown in the LB medium and were then divided into two groups. To one group, phenylalanine (0.1 mM) was added while the other group did not receive any additional phenylalanine. Samples were taken after 6.0 hours and as can be seen in the Table 6. The level of tyrosine detected in the medium increased from ˜69.0 M in control uninduced cells to ˜174 M by the IPTG induced transformants without additional phenylalanine to ˜424 M by the induced transformants which had received additional phenylalanine. Due to the absence of PAL/TAL gene, none of the cells in this study produced any PHCA during the course of the study (Table 6).

TABLE 6 Tyrosine and PHCA Production by P. aeruginosa PAH Transformants with or without Phenylalanine Addition Product Production (μM) Sample Tyrosine PHCA Control (uninduced) 68.58 0 No Phenylalanine Addition 174.22 0 0.1 mM Phenylalanine Addition 424.33 0

Example 6 Tyrosine and PHCA Production by P. aeruaginosa PAH/TAL Transformants With or Without Phenylalanine Addition

In order to study the combined effect of incorporation of the C. violaceum PAH, in addition to the endogenous P. aeruginosa PAH operon, and the modified PAL/TAL (SEQ ID NO:23) on the level of tyrosine and the PHCA formed the following experiment was performed. The P. aeruginosa was transformed with both C. violaceum PAH gene (SEQ ID NO:1) and the modified PAL/TAL gene (SEQ ID NO:23). Cells were grown in the LB medium in the presence and absence of additional phenylalanine. Table 7 summarizes the results obtained. Levels of both PHCA and tyrosine increased in induced cells that had received additional phenylalanine. When levels of tyrosine and PHCA were compared it was obvious that the rate limiting step in complete conversion of phenylalanine to PHCA via the TAL route was the PAL/TAL enzyme.

TABLE 7 Tyrosine and PHCA Production by P. aeruginosa PAH/TAL Transformants with or without Phenylalanine Addition Product Production (μM) Sample Tyrosine PHCA Control (uninduced) 40.44 0 No Phenylalanine Addition 65.66 18.34 0.1 mM Phenylalanine Addition 271.22 41.24

Example 7 Tyrosine and PHCA Production by P. aeruginosa PAH Transformants With or Without Phenylalanine Addition

In order to examine the behavior of the transformants of Example 5 while growing in the M9 and glucose medium supplemented with Mg⁺², the P. aeruginosa cells containing the C. violaceum PAH were grown in the above medium and the levels of tyrosine and PHCA were measured. The Table 8 summarizes the results obtained. The Control (uninduced) cells did not produce any tyrosine or PHCA. In the absence of additional phenylalanine induced cells produced ˜335 M of tyrosine but no PHCA When 0.1 mM phenylalanine was added, 612 M of tyrosine was produced. However, no PHCA was detected.

TABLE 8 Tyrosine and PHCA Production by P. aeruginosa PAH Transformants with or without Phenylalanine Addition Product Production (μM) Sample Tyrosine PHCA Control (uninduced) 0 0 No Phenylalanine Addition 334.88 0 0.1 mM Phenylalanine Addition 612.88 0

Example 8 Cinnamate and PHCA Production by P. aeruginosa TAL Transformants With or Without Phenylalanine Addition

The P. aeruginosa cells used in Example 5 which contained the modified PAL/TAL gene (SEQ ID NO:23) were grown in the M9 plus glucose and Mg⁺² and the levels of cinnamate and PHCA formed were measured. In all cells tested, the levels of cinnamate were higher than those observed for PHCA attesting to the presence of higher PAL than TAL activity in the modified enzyme (Table 9).

TABLE 9 Cinnamate and PHCA Production by P. aeruginosa TAL Transformants with or without Phenylalanine Addition Product Production (μM) Sample Cinnamate PHCA Control (uninduced) 86.46 40.93 No Phenylalanine Addition 132.39 129.73 0.1 mM Phenylalanine Addition 916.55 206.21

Example 9

The P. aeruginosa cells used in Example 6 which contained both C. violaceum PAH gene (SEQ ID NO:1) and the modified PAL/TAL gene (SEQ ID NO:23) were used in this experiment. They were grown in the M9 plus glucose and Mg⁺² and the levels of cinnamate and PHCA were measured after 24 hours. In all cases, higher levels of cinnamate compared to PHCA were produced (Table 10).

TABLE 10 Cinnamate and PHCA Production by P. aeruginosa PAH/TAL Transformants with or without Phenylalanine Addition Product Production (μM) Sample Cinnamate PHCA Control (uninduced) 15.82 10.34 No Phenylalanine Addition 61.54 122.97 0.1 mM Phenylalanine Addition 520.59 177.12

Example 10 Cloning and Expression of the Pseudomonas aeruginosa Phenylalanine Hydroxylase (PAH) Operon in the Phenylalanine Over-producing Escherichia Coli Phenylalanine Hydroxylase (PAH) of Pseudomonas aeruginosa: DNA Amplification and Cloning

The primers listed in the Table 11 were designed for use in incorporation of the PAH operon from P. aeruginosa into the phenylalanine over-producing E. coli strain (ATCC 31884) and the E. coli tyrosine auxotrophic strain (AT2741). The following primers of the P. aeruginosa operon designed and used for amplifying the PAH operon and its components in the E. coli hosts:

TABLE 11 Name Sequence *Re Site Gene Primer3′ 5′-GACCCAGGCGAATTCGTAA EcoRI Full SEQ ID NO:7 GGA-3′ operon Primer2′ 5′-AAAAAGCTTGCCATCACAG HindIII SEQ ID NO:8 C-3′ PhhA-5′ 5′-CGTTGCCCGGTACCTATCC KpnI Phh A SEQ ID NO:9 -3′ phhA-3′ 5′-GCGGCGGCGAAGCTTCT-3′ HindIII SEQ ID NO:10 PhhB-5′ 5′-GGACTGGTACCATGACCGC KpnI Phh B SEQ ID NO:11 ACTC-3′ phhB-3′ 5′-CCCTGGGCAAGCTTGTAGA HindIII SEQ ID NO:12 C-3′ PhhC-5′ 5′-ACCGAGTGGGGTACCGTCA KpnI Phh C SEQ ID NO:13 CCGTGA-3′ PhhC-3′ 5′-CCATCACAGCAAGCTTAGG HindIII SEQ ID NO:14 GTAAC-3′ PAHm1-5′ 5′-CCCACATGCGAATTCCAAG EcoRI PhhB/ SEQ ID NO:15 GACTC-3′ PhhC PAHm4-3′ 5′-CCATCACAGCAAGCTTAGG HindIII SEQ ID NO:16 GTAAC-3′ *Restriction site is abbreviated Re site.

The oligonucleotide primers (Table 11) were synthesized based on the deoxynucleotide sequences flanking the coding region of the P. aeruginosa phenylalanine hydroxylase gene operon described previously (Zhao G, Xia T, Song J and Jensen R A (1994) Proc. Natl. Acad. Sci., USA 91: 1366–1370). Restriction endonuclease sites (underlined sequences of the above primers of Example 1, SEQ ID NOs:5 and 6) were designed at the 5′-primer end of each primer to facilitate cloning. The P. aeruginosa genomic DNA was isolated and purified with Qiagen Kit and the DNA amplification was carried out. The amplification reaction mixture (100 μl) contained 1.0 mg of the genomic DNA template, 100 pmol each of the two primers, 2.5 units of Taq DNA polymerase(Qiagen) in 10 mM Tris-HCl, (pH 8.8), 0.2 mM each of the four dNTPs, 50 mM KCl, 1.5 mM MgCl2, and 0.01% bovine serum albumin. Thirty PCR cycles (94° C., 0.5 min; 55° C., 0.5 min and 72° C., 2.0 min) were performed. The correctly amplified DNA fragments (based on the fragment size) were cloned into a TA vector (Invitrogen, Carlsbad, Calif.) and submitted for sequence analysis. Several clones containing the gene were thus obtained.

Gene Expression and Protein Purification

To express the cloned phenylalanine hydroxlyase gene operon and its individual components, the amplified DNA fragments of interest were purified using a GeneClean II Kit (Bio 101, Inc, Carlsbad, Calif.), digested with endonucleases (Promega, Madison, Wis.) and cloned into the restriction endonuclease sites in plasmid vectors such as pD100(ATCC87222), pKSM715 (ATCC87161) and pTrc99A (Pharmacia, Piscataway, N.J.), as shown in Table 12.

TABLE 12 The Expression Plasmids used for Expression of P. aeruginosa Operon and its Components Expression Vector Selection Marker Gene pD100-phhabc Cm Full operon pD100-phha Cm Phh A pD100-phhbc Cm Phh Blphh C pTrc-phha Amp Phh A pTrc-phhb Amp Phh B pTrc-phhc Amp Phh C pTrc-phhbc Amp Phh B/phh C pKSM-phha Kan Phh A pKSM-phhb Kan Phh B pKSM-phhc Kan Phh C

The E. coli strains were transformed electroporationally with the expression vectors were grown at 37° C. in LB medium containing either 25 μg/ml of chlorophenicol for pD100 expression vectors or 100 μg/ml of ampicillin for both pKK223-3 and pTrc 19A expression vectors (Table 12) in the absence or presence of isopropyl-beta-D-thiogalactoside (IPTG).

Example 11 Growth of E. coli AT271 Tyr-auxotrophic Strain Following its Transformation with C. violaceum PAH and the Various Components of the P. aeruginosa PAH Operon

Agar plates containing M9 medium plus glucose were prepared. The tyrosine auxotrophic AT271 strain is incapable of growing on this plate in the absence of tyrosine (see Control-1). When a filter disc containing 1.0 mM tyrosine is added to the plate, growth is observed around the disc indicating the dependency of the organism to the presence of tyrosine (see Control-2). The AT271 recombinant strains used in this study included strains containing PhhA, PhhB, PhhC, PhhBC, PhhABC, PAH and PAH/PhhB/PhhC. The tyrosine disc was placed in the middle of each of the plates for these recombinants. For the strains containing PhhA, PhhB, PhhC, and PhhBC growth appeard only around the tyrosine disc indicating that these strains could not synthesize tyrosine and were still dependent on the external supply of this compound for growth. However, with recombinant strains containing PhhABC, PAH and PAH/PhhB/PhhC growth occurred on the entire plate attesting to the ability of the organism to synthesize the required tyrosine for its growth and the lack of need for the external tyrosine containing disc.

Example 12 The Effect of Iron on Growth and Tyrosine Production by Transformants of Phenylalanine Overproducing E. coli Containing the PAH Gene

In order to examine the effect of iron on the activity of PAH and therefore production of tyrosine, E. coli strains containing the PAH gene of the C. violaceum (PAH) [SEQ ID NO:1] and the PhhA component of the Pseudomonas PAH operon were grown in the M9 medium with glucose with and without addition of either FeSO₄ or Fe(NH₄)₂(SO₄)₂ (1.0 M final conc.). Samples were taken at 2.0, 6.0, 16.0, and 22 hours. The results are shown in the Table 13. Based on the results obtained, it was concluded that addition of neither of the two iron sources had any significant positive effect on the level of the tyrosine produced.

TABLE 13 Tyrosine Production of Phenylalanine Overproducing Strain (ATCC31884) Transformed with PAH from C. violaceum in the Presence and Absence of Fe⁺² Time (hour) Presence of Fe⁺² Absence of Fe⁺² 0 0 0 6 132.68 146.76 16 173.79 171.77 22 212.99 208.58

Example 13 Determination of the Levels of Phenylalanine Produced by Various Phenylalanine Overproducing E. coli Strains

The phenylalanine overproducing E. coli strains (ATCC 31882, 31883, 31884) in which inhibition of the enzyme DAHP synthase by phenylalanine, or tryptophan is removed and inhibition of the enzyme chorismate mutase P-prephenate dehydratase by phenyalanine is removed and in which enhanced levels of production of the enzymes DAHP synthase, chorismate mutase P-prephenate dehydratase and shikimate kinase are achieved (Tribe, D. E. Novel microorganism and method, U.S. Pat. No. 4,681,852, 1987), were tested for their ability to produce phenylalanine when grown in the M9 medium containing glucose. Strain ATCC 31884 produced the highest level of phenylalanine (870.54 M) as shown in Table 14.

TABLE 14 Production of Phenylalanine by Phenylalanine Overproducing Strain (ATCC 31882, 31883 and 31884) E. coli Strain Phenylalanine (μM) Control 0 31882 650.11 31883 418.23 31884 870.54

Example 14 Production of Cinnamate and Para-hydroxycinnamate by the Phenylalanine Overproducing E. coli Strains (ATCC 31884)

Experiments were performed in order to test the ability of strain ATCC 31884 transformed with the native PAL/TAL enzyme from Rhodotorula graminis to produce cinnamate and PHCA. The E. coli strain (DH5α) was used as the control and also a test strain following incorporation of the PAL/TAL enzyme. The third strain was the phenylalanine over-producer E. coli strains ATCC 31884 with the native PAL/TAL enzyme. All strains were grown in the M9 plus glucose medium and samples were taken (after 18 hrs) and prepared for HPLC analysis as described before. The results are shown in Table 15. It was interesting to note that no significant differences were observed between the levels of PHCA and cinnamate produced by strain DH5α containing the native PAL/TAL. However, much higher levels of cinnamate (˜750 M) were produced by the phenylalanine over-producing strain ATCC 31884 plus PAL/TAL compared to PHCA (175 M). These results provide yet another evidence for the higher level of PAL activity compared with TAL activity in the native PAL/TAL enzyme of Rhodotorula graminis.

TABLE 15 Production of PHCA and Cinnamate by PAL Transformants Transformant PHCA (μM) Cinnamate (μM) DH5alph (Control) 0 0 DH5α + PAL/TAL 285.23 375.33 31884 + PAL/TAL 175.55 749.74 

1. A method for the production of para-hydroxycinnamic acid comprising: a) providing a recombinant microorganism comprising: i) at least one heterologous genetic construct encoding a polypeptide having tyrosine ammonium lyase activity selected from the group consisting of SEQ ID NO:4, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22; and ii) at least one heterologous genetic construct encoding a polypeptide having phenylalanine hydroxylase activity, wherein the genetic construct is; 1) isolated from Chromobacterium violaceum and has the amino acid sequence as set forth in SEQ ID NO:2; or 2) is isolated from Pseudomonas aeruginosa and comprises at least two of the nucleic acid sequences selected from the group consisting of SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, and 15 and b) growing said recombinant microorganism in the presence of a fermentable carbon substrate whereby para-hydroxycinnamic is produced.
 2. A method according to claim 1 wherein said fermentable carbon substrate is selected from the group consisting of monosaccharides, oligosaccbarides, polysaceharides, carbon dioxide, methanol, formaldehyde, formate, and carbon-containing arnines.
 3. A method according to claim 1 wherein said fermentable carbon substrate is glucose.
 4. A method according to claim 1 wherein said genetic construct encodes a phenylalanine hydroxylase enzyme as set forth in SEQ ID NO:2.
 5. A method according to claim 1 wherein said recombinant microorganism is selected from the group consisting of bacteria, yeasts, filamentous fungi, and algae.
 6. A method according to claim 5 wherein said recombinant microorganism is selected from the group consisting of Escherichia, Salmonella Bacillus, Acinetobacter, Streptomyces, Methylobacter, Rhodococcus, Pseudomona; Cyanobacteria, Rhodobacter, Synechocystis, Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candidad, Hansenula, Debaryomyces, Mucor, Pichia, Torulopsis, Aspergillus and Arthrobotrys.
 7. A recombinant microorganism comprising: a) at least one heterologous genetic construct encoding a polypeptide having tyrosine ammonium lyase activity selected from the group consisting of SEQ ID NO:4, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22; and b) at least one heterologous genetic construct encoding a polypeptide having phenylalanine hydroxylase activity, wherein the genetic construct is 1) isolated from Chromobacterium violaceum and has the amino acid sequence as set forth in SEQ ID NO:2; or 2) isolated from Pseudomonas aeruginosa and comprises at least two of the nucleic acid seciuences selected from the group consisting of SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, and
 15. 